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| Home > Publications database > X-ray Crystallographic Study on the Mechanisms of Bacteriorhodopsin and the Sensory Rhodopsin / Transducer Complex |
| Dissertation / PhD Thesis/Book | PreJuSER-55182 |
2006
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/480
Report No.: Juel-4208
Abstract: Microbial rhodopsins belong to a family of seven-helical transmembrane retinal proteins which are found in Bacteria, Archaea and Eukaryota. They are considered to be the archetypes for ion transport and signal transduction, which use for these distinct functions a common structural design. The ion pumps Bacteriorhodopsin (BR) and Halorhodopsin (HR) operate as energy converters, whereas the photoreceptors Sensory rhodopsin I (SRI) and II (SRII) operate as light sensors providing the initial signal which via associated receptor-specific transducers (HtrI and HtrII) activate a two-component signalling cascade that moves the cell in response to light. The archaeal rhodopsins are the best understood proteins among seven-helical receptors with respect to structural information from X-ray crystallography. High resolution structures of Sensory rhodopsin II from Natronobacterium pharaonis and Bacteriorhodopsin from Halobacterium salinarum as well as Halorhodopsin have already been obtained. The X-ray structure of the complex between N.Pharaonis SRII (pSRII) and HtrII at 1.94 $\mathring{A}$ resolution was reported. In this thesis the structure of the ground state, the early K state and the signalling late M state of Sensory rhodopsin II and its cognate transducer as well as the structure of the ground state and the late M state of the Bacteriorhodopsin were investigated by means of Xray crystallography. Crystals were grown in the lipidic cubic phase which provided data to a resolution of 1.9 $\mathring{A}$ for the ground state, 2.0 $\mathring{A}$ for K state and 2.2 $\mathring{A}$ for the late M state of the Sensory rhodopsin II/transducer complex and 1.35 $\mathring{A}$ for the ground state, 1.5 $\mathring{A}$ for the late M state of Bacteriorhodopsin. The occupancies of the intermediate states trapped in the crystals at cryo temperatures were estimated from the crystallographic analysis. The structure solution based on molecular replacement yielded atomic pictures of ground, K and M state of the pSRII/transducer complex and ground and M state of bacteriorhodopsin. The refinement scheme using simulated annealing for models consisting of two conformations (one accounting for the ground state and the other for the intermediate state) with corresponding occupancies were assessed and used for structure solution. Additionally it was investigated if experimental phases obtained by isomorphous replacement and anomalous scattering would help to determine the transducer structure including linker domain. Different models of the crystal were investigated to extract structural data for the complete fold of the transducer. Results provide insights in signal transfer from pSRII to the transducer in the membrane part of the protein complex and reveal the details of the evolvement of the proton translocation channel in BR. The observed structural changes allow to propose a mechanism for the light induced activation of the complex: Upon light excitation retinal isomerization leads in K state to a rearrangement of a water cluster that partially disconnects two helices of pSRII. In the transition to late M the changes in the hydrogen bond network proceed further. The signalling state is established by tertiary structural changes induced by the new hydrogen bond pattern and the changed charge distribution. The two partially decoupled subdomains of the receptor show a relative displacement that is most significant between helices F and G which form the interface to TM2 of the transducer. The transducer responses to the receptor activation by a clockwise rotation of about 15$\circ$ of helix TM2 and a displacement of this helix by 0.9 $\mathring{A}$ at the cytoplasmic surface. The late M state structure of the wild type bacteriorhodopsin extends the knowledge of this important intermediate of the photocycle reported previously. The achieved resolution of 1.5 $\mathring{A}$ and the low twinning of the crystal enable a better definition of the model. Though this structure is very similar to the reported one it still provides new information particularly concerning the water molecule chain in the cytoplasmic part of the channel between Asp96 and Schiff base. The revealed structures allow drawing the parallels between ion transport and sensory signalling by this family of proteins. Important structural differences and similarities related to the function of these proteins are underlined.
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